Producing sgRNA-expressing lentivirus for creating chimeras with CHIME

Therapies that target the function of immune cells have significant clinical efficacy in diseases such as cancer and Although in cancer, its use in primary immune cells is limited because vector delivery is inefficient and can perturb cell states. Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages. This approach enables efficient deletion of genes of interest in major immune lineages without altering their development or function. We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8+ T cell-mediated responses to LCMV Clone 13 viral infection. These findings suggest that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo.


Introduction
Current approaches to study genes in immune cells using shRNA or CRISPR technology involve stimulating the immune cells in vitro, transducing them with a lentiviral vector, and transferring them back in vivo. These approaches are limited by inefficient vector delivery, the requirement for stimulation which can perturb cell states, and their use on populations that can be easily transferred in vivo. Here we circumvent those issues by transducing hematopoietic stem cells with a lentiviral gene-targeting sgRNA expression vector, which are then used to make bone marrow chimeras. The resulting immune system of these chimeras is deleted for the gene of interest yet is naive and Step 1: Go to NCBI gene and search for gene of interest Step 2: Find NM sequence for mRNA Step 3: Copy the "NM---" into http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design.
Step 4: Click on sgRNA Picking Results to download file. Open text file, Crtl-A, Ctrl-C and paste into new Excel document.
Step 5: Pick top 2 guides with best combined score (incorporates on and off-target effects).
Step 9: Order these oligos through IDT as lab ready.
Part 2: Guide annealing Step 1: Guides come resuspended at concentration of 100 uM.
Step 2: Prepare component for annealing reaction.

See figure in Figures section.
Step 3: Perform the reaction in thermocycler. Sequence: 37°C for 30 minutes, 95°C for 5 minutes, then goes down to 25°C at -5°C/min.

Part 3: Digestion of pXPR_053 with BsmBI
Step 1: Set up the following digest.

See figure in Figures section.
Step 2: Place the digest in an Eppendorf tube on a 55°C heat block for 1 hour.
Step 3: Prepare a 1% Agarose gel with SYBR safe DNA gel stain.
Step 4: Add DNA loading dye to some uncut pXPR_053 and some pXPR_053 from the digestion. Run these 2 samples and a 1 kB DNA marker on the gel.
Step 5: Using a UV Transilluminator determine the location of your cut gel, cut out with scalpel, and place in an Eppendorf tube.
Step 6: Perform Qiagen gel extraction kit and quantity DNA yield by Qubit.
Part 4: Ligation of annealed sgRNAs into digested pXPR_053 Step 1: Set up a ligation reaction, always set up a blank with water instead of sgRNAs.

See figure in Figures section.
Step 2: Fill up 1L beaker with water.
Step 3: Put ligation reaction tubes on floating rack and into the water beaker.
Step 4: Put beaker in 4°C cold room and incubate overnight.
Day 2: Bacteria transformation 9 a Vex+% between 1-20%. For example, assume 0.2 microliter leads to 16% Vex. (1) Low titer-This is commonly due to unhappy 293x cells or improper technique with viral supernatant regarding temperature (not keeping it cold) and resuspension (too harsh).

Anticipated Results
Generally for this protocol you should get between 30-100 million viral particles per mL.